Project Summary The goal of this project is to understand mechanisms leading to CNS viral escape in HIV-infected adults on suppressive antiretroviral therapy, particularly the role of drug resistance. Despite current antiretroviral therapies (ART), viral escape in CSF is estimated to occur in 4-20% of patients with plasma suppression, representing a barrier to HIV cure. Factors associated with CSF escape include low-level viremia, prior virologic failure, low CD4 nadir, and long duration of HIV infection, but underlying mechanisms and influence of ART drug CNS penetration, drug resistance mutations (DRMs), size of the brain reservoir, and neuroinflammation on CSF escape remain unclear. M184V/I, which confers resistance to FTC/3TC, is the most common nucleotide reverse transcriptase inhibitor (NRTI) DRM detected in blood. Preliminary studies showed M184V/I was frequently detected in patients with CSF escape despite plasma suppression, often detected together with thymidine analog resistance mutations (TAMs) in CSF. Moreover, we demonstrated a significant association between protease inhibitor (PI)-based ART regimens and CSF escape in patients with plasma suppression. Given low CNS penetration of PIs, patients with plasma M184V/I, particularly those on PI-based ART including tenofovir, have increased risk of CSF escape as a consequence of inadequate CNS penetration of ART regimens when M184V/I is present, leading to incomplete suppression of myeloid cell reservoirs in brain while selecting for TAMs and other DRMs in the CNS. To investigate these questions, we will use clinical samples and data from well-characterized cohorts followed longitudinally to characterize viral reservoirs in plasma, CSF, and brain for evidence of ongoing viral replication, profile DRMs in the RT, PR, and IN genes, and evaluate neuroinflammatory and blood-brain barrier biomarkers in patients with CSF escape. These studies will provide important insights into causes of CNS viral escape that are relevant for optimizing clinical management and overcoming obstacles to HIV cure.